We analyzed the gene expression and genetic profiles of all cell types composing normal breast tissue and in situ and invasive breast carcinomas using Serial Analysis of Gene Expression and SNP arrays, respectively. Based on these data we determined that gene expression changes occur in all cell types during breast tumor progression, while clonally selected genetic alterations were only found in cancer epithelial cells. To investigate the role of epigenetic alterations in breast tumorigenesis, we developed a novel genome-wide methylation profiling method, methylation specific digital karyotyping (MSDK). MSDK analysis of epithelial and myoepithelial cells and stromal fibroblasts isolated from normal breast tissue and in situ and invasive breast carcinomas identified cell type and tumor specific DNA methylation patterns in all three cell types. To investigate the role of myoepithelial cells and stromal fibroblasts in breast tumorigenesis we analyzed the MCFDCIS.com human xenograft model and determined that MCFDCIS.com cells are able to give raise to both epithelial and myoepithelial cells. The differentiation of these cells and the histology of the resulting xenografts are influenced by co-injected myoepithelial cells and fibroblasts: normal myoepithelial cells promote while fibroblasts inhibit the DCIS tumor phenotype. The goal of this application is to characterize the role of myoepithelial cells and stromal fibroblasts in the in situ to invasive carcinoma progression and in the differentiation of breast epithelial progenitors using in vivo and in vitro model systems, and human breast tumors. Specific aims: (1) To determine the role of epithelial-myoepithelial/fibroblast cell interactions in breast tumor progression and in the differentiation of mammary epithelial progenitor cells using human xenograft models in mice and in vitro 3D co-culture systems. (2) To evaluate the role of genes epigenetically altered in tumor myoepithelial and stromal cells in breast tumorigenesis using models described in Aim 1. Aberrantly methylated genes identified in preliminary studies will be characterized and their role in breast cancer investigated. (3) To validate the findings of Aims 1 and 2 in human breast tumors. The completion of this project will not only help us understand the role of epithelial-stromal cell interactions in breast cancer, but epigenetically altered genes and abnormally expressed paracrine factors in the tumor microenvironment may provide new targets for breast cancer treatment.